Method for rapid testing allergy

ABSTRACT

The present invention provides a method for testing an allergy capable of rapidly and highly accurately testing an allergic reaction. The method can determine whether or not a patient has an allergy or whether or not an agent that may be allergenic to a patient has an allergenicity (an allergic reactivity) in the patient. The method may comprise the steps of causing migration of leukocytes separated from a healthy human or cells of an established cell line with a chemotactic factor contained in a sample such as body fluid or blood of the patient to be tested or a sample stimulated with the agent that may be allergenic to the patient and analyzing the cell kinetics such as migration velocity, migration distance, and migration direction.

TECHNICAL FIELD

This application claims the benefit of priority of the prior Japanesepatent application (Japanese Patent Application No. 2013-025991) filedon Feb. 13, 2013, the entire contents of which are incorporated hereinby reference.

The present invention relates to a method for diagnosing an allergicdisease, a method for detecting an agent that is allergenic to orinhibits development of an allergy in a subject, a method for treatingor preventing an allergic disease by means of these methods, and a kitto be used in these methods.

BACKGROUND ART

An allergy refers to a phenomenon that an organism develops an excessiveimmune response to a specific allergenic agent, namely, an antigendesignated as an allergen. A variety of foreign substances derived fromthe environment to which an organism is exposed under its livingenvironment can become an allergen (an allergenic agent) that causes anallergy. Typical allergic diseases induced by various foreignsubstances, such as pollen, foods, body compositions or dejections ofanimals, molds, chemicals and drugs, include allergic rhinitis (pollenallergies), allergic conjunctivitis, food allergies, and drug allergies.

These allergic diseases develop when any of the aforementionedallergenic agents, which works as an allergen by itself or does not workas an antigen by itself but is changed to an allergen by binding to amacromolecule such as a protein in the body, elicits an excessive immuneresponse causing an allergy.

Conditions caused by allergies, particularly by drug allergies, includerelatively mild drug rashes and blood disorders, severe drug rashesknown as Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis(TEN) and drug-induced hypersensitivity syndrome (DINS), as well asserious conditions causing sequela or death at the worst, such as organdysfunction of, for example, liver, lung or digestive tube, andanaphylactic shock.

Drug withdrawal due to the development of a drug allergy, which meansinterruption of the treatment of the primary disease, should be avoidedby preventing the allergy.

However, means for preventing allergic diseases are limited. Response toa drug is greatly different among individuals and may be influenced bymultiple factors including concomitant drugs. Among currently performedallergy tests, an intradermal test or patch test performed in advance toadministering a drug cannot always accurately predict development of anallergy to the drug. A drug challenge test is undesirable because itimposes a large burden on the patient and increases risk of shock if anallergy is induced.

Accordingly, once a drug allergy develops, careful and rapid detectionand determination of the causative agent is desired.

The causative agent for a drug allergy is currently detected ordetermined by, for example, a drug-induced lymphocyte stimulation test(DLST) or a leukocyte migration test (LMT). In addition, atime-consuming drug elimination method in which a suspected drug iswithdrawn may be employed.

The DLST and the LMT are useful, in view of safety, because bloodcollected from the patient is tested. The patient is not involved afterthe blood collection. Disadvantages of the tests include that the testsneed approximately 10 ml of blood for each drug to be tested, requireabout one week for obtaining the result and could give false positivereactions.

A drug allergy can be caused by any of allergic mechanisms includingtypes I to IV. For planning treatment and relief of a symptom presentedby a patient, it is important to establish a definitive diagnosis thatthe symptom is a drug allergy through an immune response or the symptomis a non-allergic adverse reaction (pseudo reaction) involving no immuneresponse.

In a blood test for diagnosing an allergy, indicators such aseosinophils and IgEs are generally measured. Though many of suchindicators can be used or referred to for diagnosing an allergic statefor example in type I allergy, they are not universal indicators andcannot be used for the definite diagnosis of all types of allergies.

In addition, positive rates are not very high in tests such as the DLST(Non Patent Literatures 1 and 2). One of the reasons is probably that itfails to exclude pseudo reactions different from drug allergies.

On the other hand, it has been revealed that a specific humanhistocompatibility antigen (HLA) genotype is a risk factor of severe SJSor TEN developed after taking allopurinol, a gout suppressant, orcarbamazepine, an antiepileptic drug in south Asians, particularlyChinese people (Non Patent Literatures 3 and 4). Methods for predictinga risk of a drug based on the HLA gene has been developed (Non PatentLiterature 5). However, this method is used only for avoiding the riskand is not sufficient.

Accordingly, there is a demand for a method which can test a drugallergy accurately, and a method which can efficiently and sensitivelytest or determine a drug that could induce a drug allergy, for thepurpose of selecting an alternative drug for treating the primarydisease in a patient.

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Mika Muto et al., the Japanese Journal of    Dermatology, 2000; 110(10): 1543-1548-   Non Patent Literature 2: Suzuki Y, et al., CHEST 2008; 134(5):    1027-1032-   Non Patent Literature 3: Somkrua R, et al., BMC Med Genet, 2011; 12:    118-   Non Patent Literature 4: Yip V L, et al., Clin Pharmacol Ther. 2012;    92(6): 757-65-   Non Patent Literature 5: MediBiC Group, Aug. 27, 2012, press release

SUMMARY OF INVENTION

As a result of earnest studies, the present inventors have found thatwhether or not a subject has an allergy can be rapidly, accurately andefficiently tested by using serum of the subject having an allergy-likesymptom and cells such as leukocytes of a healthy volunteer. The presentinventors also have found that whether or not an allergenic agentdevelops an allergy in a subject can be tested by using a reactionsolution or culture supernatant obtained by stimulating lymphocytes ofthe subject with the allergenic agent, and cells such as leukocytes of ahealthy volunteer. On the basis of these findings, the inventors haveaccomplished a very useful and simple method for testing an allergy in asubject, which can be used for identifying an agent that is allergenicto the subject or a method for predicting the risk that the subjectdevelops an allergy.

In one aspect, the present invention provides a method for diagnosing anallergic disease in a subject or determining a risk that a subjectdevelops an allergy, comprising the steps of:

(1) causing migration of chemotactic cells with a sample derived fromthe subject; and

(2) measuring the cell kinetics (for example, the migration distanceand/or migration velocity) of the migrating cells.

In another aspect, the present invention provides a method fordetermining, detecting or quantifying an agent that is allergenic to orinhibits development of an allergy in an animal subject, comprising thesteps of:

(1) culturing leukocyte cells derived from the subject in a mediumcontaining a sample;

(2) collecting the culture supernatant;

(3) causing migration of chemotactic cells with the culture supernatant;and

(4) measuring the cell kinetics (for example, the migration distanceand/or migration velocity) of the migrating cells,

wherein the method is

for determining whether or not the sample is allergenic to or inhibitsdevelopment of an allergy in the subject,

for detecting an agent that is allergenic to or inhibits development ofan allergy in the subject in the sample, or

for quantifying an agent that is allergenic to or inhibits developmentof an allergy in the subject in the sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows microscopic images of the chemotactic test. Chemotacticcells, i.e. leukocytes were added to the lower well of a chemotacticchamber. Serum derived from a healthy volunteer, a volunteer with mildcutaneous pruritus or a patient suspected of having a drug allergy wasadded to the upper well of the chamber. The migration of the cells atthe addition of the serum and 20 minutes after the addition are shown.Cell kinetics was observed every 2 minutes starting from the addition ofthe serum and recorded for 20 minutes.

FIG. 2A shows the spatially coordinated migration of the individualcells in reference to the original place where the cells were at thetime of starting the observation.

FIG. 2B shows analytical results of the migration velocity.

FIG. 2C shows analytical results of the migration distance.

FIG. 3A shows percentages of the migration velocity relative to that ofthe control group (the serum of the healthy volunteer: FIG. 1a ).

FIG. 3B shows percentages of the migration distance relative to that ofthe control group (the serum of the healthy volunteer: FIG. 1a ).

FIG. 4A shows microscopic images of chemotactic test using Jurkat cellsin a chemotactic chamber. Serum of #3 patient was added to the upperwell of the chamber. The images at the addition of the serum and that at50 minutes after the addition are shown. The cell kinetics, i.e.migration velocity, was observed and recorded every two minutes for 50minutes after the addition of the serum.

FIG. 4B shows microscopic images of chemotactic test using Jurkat cellsin a chemotactic chamber. Serum of a healthy volunteer was added to theupper well of the chamber. The images at the addition of the serum andthat 50 minutes after the addition are shown. The cell kinetics, i.e.migration velocity, was observed and recorded every two minutes for 50minutes after the addition of the serum.

FIG. 4C shows analytical results of the migration distance caused by theserum of patient #3.

FIG. 4D shows analytical results of the migration distance caused by theserum of the healthy volunteer.

FIG. 4E shows analytical results of the migration velocity.

FIG. 5A shows analytical results of chemotaxis or migration abilitytoward phytohemagglutinin (hereinafter also designated as PHA) (a) as acontrol, gasmotin (b), other suspected drugs (c and d), and a solvent(DMSO, e).

FIG. 5B shows analytical results of the migration velocity.

FIG. 5C shows analytical results of the migration distance.

FIG. 6A shows percentages of the migration velocity relative to that ofthe control group (the reactive solution stimulated with PHA: a).

FIG. 6B shows percentages of the migration distance relative to that ofthe control group (the reactive solution stimulated with PHA: a).

FIG. 7A shows analytical results of chemotaxis or migration abilitytoward a cedar pollen antigen SBP.

FIG. 7B shows analytical results of the migration velocity.

FIG. 7C shows analytical results of the migration distance.

FIG. 8A shows percentages of the migration velocity relative to that ofthe control group (the cedar pollen antigen SBP: 0 μg/ml).

FIG. 8B shows percentages of the migration distance relative to that ofthe control group (the cedar pollen antigen SBP: 0 μg/ml).

FIG. 9 shows increase of the cell kinetics depending on the pollenextract concentration.

FIG. 10A shows influence of Hinyosen on the cell kinetics.

FIG. 10B shows influence of each component of the Kampo product on thecell kinetics.

FIG. 11 shows atypical negative test results of the method provided bythe present invention. The patient did not have any serious and typicalallergy-like skin symptom.

FIG. 12 shows typical positive test results of the method provided bythe present invention. The patient had serious and typical allergy-likeskin symptoms.

FIG. 13 shows increase of migration distance of HL-60 cells caused bythe culture supernatant of mononuclear cells obtained from a patienthaving cedar pollen allergy and exposed to a pollen extract.

DESCRIPTION OF EMBODIMENTS

An allergic disease herein may be caused by, and an allergy herein maybe, a type 1, II, III or IV allergy, such as hives, a food allergy, apollen allergy, atopic dermatitis, an allergy caused by a chemical, oran allergy caused by a drug. Examples of conditions caused by a drugallergy include drug rashes such as Stevens-Johnson syndrome (SJS),toxic epidermal necrolysis (TEN) and drug-induced hypersensitivitysyndrome (DINS). Examples of the drugs that induce an allergy includeantibiotics such as penicillins, cephems, aminoglycosides, macrolides,and quinolones, nonsteroidal anti-inflammatory drugs, pyrine drugs(antipyretic analgesics), local anesthetics, iodine-based drugs, centralnervous system drugs such as carbamazepine, and gout suppressants suchas allopurinol.

The sample used in an aspect of the present invention may be abiological component derived from an animal subject, such as body fluidor blood, and more specifically, its component such as lymph, serum orplasma. The sample used in another aspect of the present invention maybe a reactive solution or culture supernatant in which leukocytes froman animal subject were cultured with a candidate agent that may beallergenic to or inhibit development of an allergy in the subject, aswell as the candidate agent itself. The amount of the sample used in theaspects of the present invention may be appropriately determined by aperson skilled in the art. In one embodiment, the sample may be a verysmall amount (for example, 1 to 10 μl) of the serum, or a very smallamount (for example, 1 ng to 1 μg) of the candidate agent.

The animal subject herein is a mammal, preferably a human.

For example, in the method for diagnosing an allergic disease in asubject or determining a risk that a subject develops an allergyprovided by the present invention, the sample may be a biologicalcomponent of the subject, such as body fluid or blood, morespecifically, its component such as lymph or serum. For example, in themethod for determining, detecting or quantifying an agent that isallergenic to or inhibits development of an allergy in a subject, thesample may be a reaction solution or culture supernatant obtained byadding an agent that may be allergenic to or may inhibit development ofan allergy in the subject to a culture medium comprising leukocytesderived from the subject and stimulating the leukocytes with the agent.

The allergenic agent can be an agent that works as an allergen byitself, or does not work as an antigen by itself but is changed to anallergen by binding to a macromolecule such as a protein in a livingbody, and may elicit an excessive immune response which causes anallergy in a subject. Typical examples of the allergenic agent includepollen, chemicals, drugs and foods.

The allergy inhibitor is an agent that suppresses development of anallergy in a subject.

The term “chemotaxis” as used herein, which is also mentioned as“migration ability” or “chemical taxis”, means a property of an organismincluding a cell to make a directional migration according to aconcentration gradient of a specific chemical substance.

The term “chemotactic factor” as used herein, which is also mentioned as“migration factor”, refers to a chemical substance causing thechemotaxis defined above. Typical examples of the chemotactic factorinclude interleukins, cytokines, and chemokines.

The chemotactic cells herein may be cells having the chemotaxis ormigration ability. Examples include leukocytes and established celllines. The leukocytes may be granulocytes such as neutrophils,eosinophils or basophils, mononuclear cells such as monocytes orlymphocytes, or a mixture thereof.

The chemotactic cells used herein for determining whether or not asubject has an allergy or whether or not an agent develops an allergy ina subject is, for example, leukocytes of a healthy volunteer,particularly leukocytes including granulocytes, which can be simplyseparated and can respond to all humoral factors (chemotactic factors).Purified and separated neutrophils or eosinophils, or cells of anestablished cell line expressing a specific receptor may also be used.The use of an established cell line eliminates the step of cellpreparation through separation and purification processes and makes thetest procedures simple. Examples of the established cell line includehuman leukocytic cell lines such as human T-cell cell lines (forexample, Jurkat cells) and human promyelocytic cell lines (for example,HL-60 cells). Other examples of the human leukocytic cell line include,but are not limited to, HL60RG, K562, MOLT-4F, IM-9, CCRF-HSB2, CCRF-SB,CCRF-CEM, RPMI 8226, RPMI 1788, HLCL-1, HEL, KG-1, BALL-1, TALL-1,P31/FUJ, P30/OHK, P32/ISH, A4/Fuk, A3/KAW, KU812, KU812E, KU812F, KY821,KY821A3, THP-1, LC4-1, SCC-3, KMS-12-BM, KMS-12-PE, PEER, RAJI, U937,CCRF-CEM, MOLT-4, MOLT-3, KG-1, HS-Sultan, WIL2-NS, Saudi, Ramos (RA1),U937 cl1-14, U937 cl1-22, KO52, KHM-1B, SKM-1, MLMA, JKT-beta-del,KHYG-1, TK, MY-M12, MY-M13, SLVL, MY, RPMI 8226, RPMI 1788, NC-37,Namalwa, Raji, MEG-01, MEG-01SSF, MOLT-4, KU812, CCRF-CEM, CMK-86,CMK-11-5, MEG-01s, NOMO-1, NOMO-1s, NKM-1, MEG-A2, NAGL-1, MTA, TMD5,KAI3, Kasumi-4, HL60(S), KHM-10B, K562/ADM, Kasumi-1, Kasumi-3,Kasumi-6, Ki-JK, CPT-K5, NOMO-1/ADM, NCO2, SKNO-1, KMS-11, KMM-1,KMS-21BM, KMS-26, KMS-27, KMS-28PE, KMS-28BM, KMS-34, KMS-20, TK,KCL-22, PL-21, MKPL-1, NALL-1, RC-K8, HD-70, DL-40, delta-47, PALL-2,FLAM-76, B104, KML-1, STR-428, KMS-33, KHM-2B, Kasumi-2, Kasumi-5,Kasumi-7, Kasumi-8, Kasumi-9, Kasumi-10, Minami-1, Minami-2,KasumiA-541, KasumiA-568, and KasumiA-554.

The cells can be cultured appropriately by a person skilled in the art.For example, the Jurkat cells can be cultured in a DMEM containing 10%FBS and the leukocytes and the mononuclear cells can be cultured in aRPMI 1640 containing 10% FBS.

Migration of the chemotactic cells can be caused by creating aconcentration gradient of a chemotactic factor and placing the cellsunder the concentration gradient. A concentration gradient of achemotactic factor can be appropriately created by a person skilled inthe art. It can be created by using, for example, a chemotaxis chambersuch as a Dunn chamber or a μ-Slide. Alternatively, a concentrationgradient of a chemotactic factor can be created by creating aconcentration gradient of a sample or a culture supernatant containingthe chemotactic factor. In one embodiment, on the basis of cell kinetics(for example, migration distance, migration time and/or migrationdirection) of chemotactic cells in the presence of a concentrationgradient of a chemotactic factor contained in a sample, an allergy in asubject is diagnosed, a risk that a subject develops an allergy isdetermined, and an agent that is allergenic to or inhibits developmentof an allergy in an animal subject is determined, detected orquantified.

The cell kinetics herein means motility of cells such as chemotaxis ormigration ability, and specifically means migration distance, migrationtime, or migration direction of the cells. The cell kinetics can bemeasured by observing each cell in a coordinate space in a field of amicroscope and sequentially recording the tracks of the migration.

In one embodiment, the methods provided by the present invention can becarried out rapidly, sensitively and/or specifically with a very smallamount of a sample. In one embodiment, the amount of blood of a subjectnecessary for the methods provided by the present invention may besmaller than that necessary for the DLST or the LMT which requires about10 ml of blood for each test agent. For example, according to thisembodiment, the required amount of blood (cells) of a subject may be 100μl or less for each test agent. In one embodiment, the methods providedby the present invention can provide a result more quickly than the DLSTor the LMT which generally takes about 80 hours. For example, accordingto the present invention, all the steps can be completed in about 2hours in the diagnosis method using serum or plasma, and in about hoursin the other methods. In one embodiment, the method for diagnosing anallergy in a subject provided by the present invention can determine theseverity of the allergic symptom in the subject. For example, asdescribed in Example 1, the method can distinguish a patient having amild allergic symptom from a patient having a severe allergic symptom.For example, as described in Example 5, when a risk that an allergenicagent causes an allergy in an animal subject (preferably a human) isdetermined, the possible severity of the allergy to be developed by theagent can be determined.

A person skilled in the art can use an appropriate measuring system formeasuring cell kinetics (for example, migration distance and/ormigration velocity) of migrating cells. For example, a system thatenables a person skilled in the art to sequentially observe and recordcell kinetics of each cell in the presence of a concentration gradientof a chemotactic factor contained in a test liquid (serum) in anappropriate coordinate space within a field of a microscope, for examplea chemotaxis chamber such as a Dunn chamber or a μ-Slide, may be used. Asystem which is not integrated with a microscope may also be used aslong as cell kinetics of each cell can be sequentially observed andrecorded.

In one embodiment, cell kinetics (for example, migration distance and/ormigration velocity) of migrating cells can be measured by processingimages which are obtained by sequentially observing and recording thecell kinetics of each chemotactic cell with an image processingsoftware, for example those provided free of charge or commerciallyavailable, and calculating various parameters concerned with chemotaxisor migration ability of the cells such as time, distance and directionof the migration.

According to the method provided by the present invention, whether ornot an animal subject (preferably a human subject) has an allergicdisease, whether or not an animal subject has a risk of developing anallergy, whether or not a sample is an agent that is allergenic to ananimal subject, whether or not a sample contains an agent that isallergenic to a an animal subject, whether or not a sample is an agentthat inhibits an allergy in a subject, or whether or not a samplecontains an agent that inhibits an allergy in a subject can bedetermined on the basis of data of the cell kinetics (for example,migration distance and/or migration velocity) of the migrating cells.

For example, in the method for diagnosing an allergic disease in ananimal subject or determining a risk that an animal subject develops anallergy provided by the present invention comprising the steps of:

(1) causing migration of chemotactic cells with a sample derived fromthe subject; and

(2) measuring the cell kinetics (for example, the migration distanceand/or migration velocity) of the migrating cells,

whether or not the subject has an allergic disease or whether or not thesubject has a risk of developing an allergy may be determined by testinga negative control and/or a positive control in addition to the samplederived from the subject. Examples of the negative control include, butare not limited to, samples (for example, serum) derived from a healthyanimal (preferably a human) or an animal (preferably a human) not havingan allergic disease. Examples of the positive control include a culturesupernatant obtained by stimulating leukocyte cells (for example,mononuclear cells) with a leukocyte stimulator. Such a culturesupernatant can be prepared, for example, by adding a leukocytestimulator to a culture medium of leukocyte cells and culturing thecells for a period of time (for example, for one day, two days, threedays, four days or five days). Examples of the leukocyte stimulatorinclude a mitogen. Specific examples of the leukocyte stimulatorsinclude, but are not limited to, plant lectins such as PHA, concanavalinA and PWM (pokeweed mitogen), LPS (lipopolysaccharide), PPD (purifiedprotein derivative of tuberculin), and dextran sulfate.

For example, when migration distance of migrating cells caused by asample (for example, serum) derived from an animal subject (preferably ahuman) are longer than migration distance of migrating cells caused by asample (for example, serum) derived from a healthy animal (preferably ahuman) or an animal (preferably a human) not having an allergic diseaseas a negative control, the animal subject (preferably the human) can bedetermined to have an allergic disease or a risk of developing anallergy. In one embodiment, when migration distance of migrating cellscaused by a sample (for example, serum) derived from an animal subject(preferably a human) are 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2times or more longer than that caused by a sample (for example, serum)derived from a healthy animal (preferably a human) or an animal(preferably a human) not having an allergic disease, the animal subject(preferably the human) can be determined to have an allergic disease ora risk of developing an allergy.

Alternatively, when migration velocity of migrating cells caused by asample (for example, serum) derived from an animal subject (preferably ahuman) is higher, for example, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or2 times or more higher than that caused by a sample (for example, serum)derived from a healthy animal (preferably a human) or an animal(preferably a human) not having an allergic disease, the animal subject(preferably the human) can be determined to have an allergic disease ora risk of developing an allergy.

On the other hand, when value of migration distance and/or migrationvelocity of migrating cells caused by a sample (for example, serum)derived from an animal subject (preferably a human) is 1.1, 1, 0.9 or0.8 times as or less than that caused by a sample (for example, serum)derived from a healthy animal (preferably a human) or an animal(preferably a human) not having an allergic disease, the subject can bedetermined not to have an allergic disease or a risk of developing anallergy.

Furthermore, when value of migration distance and/or migration velocityof migrating cells caused by a sample (for example, serum) derived froman animal subject (preferably, a human) is equivalent to or larger, forexample, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 times ormore larger than that caused by a culture supernatant of leukocyte cellsstimulated with PHA as a positive control, the animal subject can bedetermined to have an allergic disease or a risk of developing anallergy.

In the method for diagnosing an allergic disease in a subject or fordetermining a risk that a subject develops an allergy provided by thepresent invention, the positive control or the negative control asdescribed above can be used alone or in combination.

In one embodiment, the method for diagnosing an allergic disease in asubject or for determining a risk that a subject develops an allergyprovided by the present invention can be employed for determining thedegree of the allergic disease or the degree of the risk. For example,the degree of an allergic disease in a subject or the degree of a riskthat a subject develops an allergy may be determined by generating acalibration curve on the basis of migration distance and/or migrationvelocity of a given amount of migrating cells caused with a plurality ofknown amounts of a positive control (for example, culture supernatantsprepared by stimulating leukocyte cells such as mononuclear cells withdifferent concentrations of a leukocyte stimulator such as PHA), andquantifying a test result of a sample derived from an animal subject(preferably, a human) with the calibration curve.

In one embodiment, time-dependent change of the degree of an allergicdisease in an animal subject or the degree of a risk that an animalsubject develops an allergy may be determined by collecting samples fromthe animal subject (preferably, a human) at different time points,testing the samples by the method for diagnosing an allergic disease ina subject or for determining a risk that a subject develops an allergyprovided by the present invention, and comparing migration distanceand/or migration velocity of migrating cells caused by the collectedsamples at each time. For example, if value of migration distance and/ormigration velocity of migrating cells is larger than that caused by thesample previously collected from the same animal subject (preferably,the same human), it can be determined that the allergic symptom hasbecome worse, or that the risk that the subject develops the allergy hasbeen increased. On the other hand, for example, if value of migrationdistance and/or migration velocity of migrating cells is smaller thanthat caused by the sample previously collected from the same animalsubject (preferably, the same human), it can be determined that theallergic symptom has been ameliorated or that the risk that the subjectdevelops the allergy has been reduced.

In one embodiment, the method for diagnosing an allergic diseaseprovided by the present invention can be utilized for a differentialdiagnosis of an allergic disease from a non-allergic disease. Forexample, when serum derived from a subject suspected of having drugrashes and serum derived from a healthy human or from a patient of anon-allergic disease are tested and migration distance and/or migrationvelocity of migrating cells measured for the serum derived from thesubject is longer and/or higher than that measured for the serum derivedfrom the healthy human or the patient of the non-allergic disease, thesubject is diagnosed as having an allergic disease.

In one embodiment, whether or not an animal subject has an allergicdisease or has a risk of developing an allergy can be determined bymeasuring migration distance of chemotactic cells during a fixed timeperiod and classifying the result in four classes of: negative (−), noor substantially no chemotactic factor in the sample; suspected (+/−), achemotactic factor is suspected to be present; positive (+), achemotactic factor is present; and strongly positive (++), a chemotacticfactor is present in a higher concentration.

In the method provided by the present invention for determining ordetecting an agent that is allergenic to or inhibits development of anallergy in an animal subject comprising the steps of:

(1) culturing leukocyte cells (for example, mononuclear cells) derivedfrom the animal subject in a medium containing a sample;

(2) collecting the culture supernatant;

(3) causing migration of chemotactic cells with the culture supernatant;and

(4) measuring the cell kinetics (for example, the migration distanceand/or migration velocity) of the migrating cells,

leukocyte cells (for example, mononuclear cells) derived from a healthyanimal may be used in place of leukocyte cells (for example, mononuclearcells) derived from the animal subject, for determining whether or notthe sample is an agent that is allergenic to or inhibits development ofan allergy in the animal subject, or whether or not the sample containsan agent that is allergenic to or inhibits development of an allergy inthe animal subject. A positive control may be tested as well as thesample, for determining whether or not the sample is an agent that isallergenic to or inhibits development of an allergy in the animalsubject, or whether or not the sample contains an agent that isallergenic to or inhibits development of an allergy in the animalsubject. Examples of the positive control include a leukocyte stimulatorsuch as a mitogen. Specific examples of the positive control include,but are not limited to, plant lectins such as PHA, concanavalin A andPWM, LPS, PPD, and dextran sulfate.

For example, when leukocyte cells (for example, mononuclear cells)derived from a healthy animal (preferably, a human) is used in place ofleukocyte cells (for example, mononuclear cells) derived from an animalsubject (preferably, a human) and value of migration distance and/ormigration velocity of migrating cells obtained with the leukocyte cells(for example, the mononuclear cells) derived from the animal subject(preferably, the human) is larger, for example, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9 or 2 times or more larger than that obtained with theleukocyte cells (for example, the mononuclear cells) derived from thehealthy animal (preferably, the human), it can be determined that thesample is or contains an agent that is allergenic to the animal subject.

Furthermore, if the value of migration distance and/or migrationvelocity of migrating cells caused by PHA as a positive control isequivalent to or larger, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9 or 2 times or more larger than that obtained for thesample, it can be determined that the sample is or contains an agentthat is allergenic to the animal subject.

For example, if the value of migration distance and/or migrationvelocity of migrating cells obtained for a mixture containing PHA and asample is smaller, for example, 0.5 times or less smaller than thatobtained for PHA, it is determined that the sample is or contains anagent that inhibits development of an allergy in the animal subject.

In the method for determining or detecting an agent that is allergenicto or inhibits development of an allergy in an animal subject providedby the present invention, leukocyte cells (for example, mononuclearcells) derived from a healthy animal (preferably, a human) may be usedin place of leukocyte cells (for example, mononuclear cells) derivedfrom the animal subject (preferably, the human subject), and/or apositive control may be further tested in addition to the sample.

In one embodiment, it can be determined that a sample is or contains anagent that is allergenic to or inhibits development of an allergy in theanimal subject by measuring migration time necessary for chemotacticcells migrating through a fixed distance and classifying the results infour classes of: negative (−), no or substantially no chemotactic factorin the sample; suspected (+/−), a chemotactic factor is suspected to bepresent; positive (+), a chemotactic factor is present; and stronglypositive (++), a chemotactic factor is present in a higherconcentration.

In one embodiment, a chemotactic factor is released from leukocyte cellsderived from a subject in proportion to the concentration of anallergenic agent or an allergy inhibitor and chemotactic cells migrates.Accordingly, when determining whether or not an agent that is allergenicto the animal subject is contained in the sample, the sample may beserially diluted and the concentration-dependent change of the cellkinetics (for example, the migration distance and/or migration velocity)of the chemotactic cells may be detected.

In the method provided by the present invention for quantifying an agentthat is allergenic to or inhibits development of an allergy in an animalsubject in a sample comprising the steps of:

(1) culturing leukocyte cells (for example, mononuclear cells) derivedfrom the animal subject in a medium containing a known amount of theagent that is allergenic to the subject, a known amount of the agentthat inhibits development of an allergy in the subject, or the sample;(2) collecting the culture supernatant;(3) causing migration of chemotactic cells with the culture supernatant;and(4) measuring the cell kinetics (for example, the migration distanceand/or migration velocity) of the migrating cells, the agent that isallergenic to or inhibits development of an allergy in the subject inthe sample can be quantified by comparing the cell kinetics (forexample, the migration distance and/or migration velocity) of themigrating cells measured in the presence of the known amount of theagent that is allergenic to the subject or a known amount of the agentthat inhibits development of an allergy in the subject with that of themigrating cells measured in the presence of the sample.

In one embodiment, an agent that is allergenic to or inhibitsdevelopment of an allergy in the subject can be quantified with acalibration curve of the migration distance and/or migration velocitythat is generated by testing a plurality of known concentrations of theagent that is allergenic to or inhibits development of an allergy in thesubject.

The method for determining a risk that a subject develops an allergyprovided by the present invention can be used for predicting whether ornot a test agent induces an allergy in the subject. Accordingly, in oneembodiment, the method for determining a risk that a subject develops anallergy provided by the present invention is applicable as a novelmethod for predicting the development of the allergy in the subject andis extremely useful.

If the method for determining a risk that a subject develops an allergyprovided by the present invention is utilized as the method forpredicting the development of the allergy in the subject, a necessaryamount of a sample is smaller than that in the DLST or the LMT, in whichabout 10 ml of a sample is necessary for each drug to be tested. Forexample, the required amount of blood (cells) of a subject may be 100 μlor less for each drug to be tested. This property of the method, thatis, it does not require blood or cells of a subject as much as thatrequired in the DLST or the LMT, is very advantageous since the burdenon the subject is reduced.

In one embodiment, the methods provided by the present inventioncomprises the steps of:

preparing a sample containing serum of the subject or a samplecontaining a chemotactic factor generated by exposing leukocytes of thesubject to a test agent such as a test chemical, a test drug or a testfood that may be allergenic to or may inhibit the development of anallergy in the subject;

preparing chemotactic cells from, for example, blood of the subject or ahealthy human;

measuring the cell kinetics of the cells;

processing an image of the cell kinetics; and,

determining whether or not the subject has an allergic disease, whetheror not the subject has a risk of developing an allergy, whether or notthe test agent is an agent that is allergenic to or inhibits developmentof an allergy in the subject, or whether or not the test agent containsan agent that is allergenic to or inhibits development of an allergy inthe subject on the basis of the processed image.

In the step of preparing the sample, the sample suitable to an allergytest is prepared by a conventional method from a biological componentsuch as body fluid or blood of the subject. For example, if serum isused as the sample, the sample can be prepared by adding a serumseparating agent to blood collected from the subject and centrifugingthe mixture. Other samples can be prepared similarly by a conventionalmethod.

When the methods provided by the present invention is used fordetermining or testing whether or not an agent is allergenic to orinhibits development of an allergy in the subject, or for predictingwhether or not an agent induces an allergy in the subject, the sample tobe tested may be a solution containing a chemotactic factor that issecreted from leukocytes exposed to the agent to be tested.

In the step of preparing the chemotactic cells, the chemotactic cellscan be prepared, for example, by adding an agent such as heparin or EDTAto blood from a healthy volunteer having no allergic symptom, separatingleukocytes including granulocytes such as neutrophils with densitygradient centrifugation in a conventional manner, and suspending theseparated leukocytes in an appropriate culture medium. Alternatively,cells of an established cell line expressing a specific receptor can beused as the chemotactic cells. Such chemotactic cells can be prepared bysuspending the cells of the cell line in an appropriate culture medium.

In the step of measuring the cell kinetics, for example, a fixed amountof the chemotactic cells is injected into the lower well of acommercially available holder for testing chemotactic cells and suckedup to the end of an observation terrace, and a fixed amount of thesample such as serum is injected into the upper well to generate aconcentration gradient of the sample, and an image of the chemotacticcells formed on the observation terrace is measured.

In the step of processing the image, the image of the chemotactic cellsformed in the step of measuring the cell kinetics is analyzed with animage processing software and various parameters such as migrationdistance, migration time and migration direction of the chemotacticcells in the presence of the sample such as serum are calculated.

In the step of the determination, the presence or absence of an allergicreaction in the subject and the degree thereof, which is caused by anagent that is allergenic to or inhibits development of an allergy in thesubject in a sample such as serum, can be determined by comparingvarious parameters obtained in the step of processing an image withpredetermined criteria. Similarly, whether or not the subject has anallergic disease or whether or not the subject has a risk of developingan allergy can be determined, or an agent that is allergenic to orinhibits development of an allergy in the subject is determined,detected or quantified.

In one embodiment, the methods provided by the present invention arecarried out in vitro. A result obtained by the method for determining arisk that a subject develops an allergy or the method for determining,detecting or quantifying an agent that is allergenic to or inhibitsdevelopment of an allergy in a subject provided by the present inventioncan assist a doctor to diagnose an allergic disease.

An allergy can be treated or prevented according to a result of themethod for diagnosing an allergy in a subject, the method fordetermining a risk that a subject develops an allergy, or the method fordetermining, detecting or quantifying an agent that is allergenic to asubject provided by the present invention. For example, if an agent thatis allergenic to a subject is determined by the method for determining,detecting or quantifying an agent that is allergenic to an animalsubject provided by the present invention, the allergy can be treated orprevented by reducing the contact of the subject with the agent.Alternatively, for example, if a subject is determined to have anallergic disease or not to have an allergic disease but have a high riskof developing an allergic disease by the method for diagnosing anallergy in a subject or the method for determining a risk that a subjectdevelops an allergy provided by the present invention, the allergy canbe treated or prevented by administering an anti-allergic agent (forexample, an antihistamine). Accordingly, in one embodiment, the presentinvention provides a method for treating or preventing an allergy in asubject comprising carrying out the method for diagnosing an allergy ina subject, the method for determining a risk that a subject develops anallergy, or the method for determining, detecting or quantifying anagent that is allergenic to a subject provided by the present invention.In one embodiment, the method for treating or preventing an allergy mayfurther comprise the step of reducing a contact of the subject with theagent that is allergenic to the subject and/or the step of administeringan anti-allergic agent to the subject. For example, if the symptom ofthe allergy is drug rashes, the method may comprise the step of stoppingadministration of the causative drug.

In one embodiment, the present invention provides a kit comprising achemotaxis chamber, leukocytes or an established cell line, and a cellculture medium for diagnosing an allergic disease in a subject; fordetermining a risk that a subject develops an allergy; or fordetermining, detecting or quantifying an agent that is allergenic to orinhibits development of an allergy in a subject. The leukocytes can beprepared from, for example, blood of a healthy human. Examples of theestablished cell line include human leukocytic cell lines such as humanT-cell cell lines (for example, Jurkat cells) and human promyelocyticcell lines (for example, HL-60 cells). Other examples of the humanleukocytic cell line include, but are not limited to, HL60RG, K562,MOLT-4F, IM-9, CCRF-HSB2, CCRF-SB, CCRF-CEM, RPMI 8226, RPMI 1788,HLCL-1, HEL, KG-1, BALL-1, TALL-1, P31/FUJ, P30/OHK, P32/ISH, A4/Fuk,A3/KAW, KU812, KU812E, KU812F, KY821, KY821A3, THP-1, LC4-1, SCC-3,KMS-12-BM, KMS-12-PE, PEER, RAJI, U937, CCRF-CEM, MOLT-4, MOLT-3, KG-1,HS-Sultan, WIL2-NS, Daudi, Ramos (RA1), U937 cl1-14, U937 cl1-22, KO52,KHM-1B, SKM-1, MLMA, JKT-beta-del, KHYG-1, TK, MY-M12, MY-M13, SLVL, MY,RPMI 8226, RPMI 1788, NC-37, Namalwa, Raji, MEG-01, MEG-01SSF, MOLT-4,KU812, CCRF-CEM, CMK-86, CMK-11-5, MEG-01s, NOMO-1, NOMO-1s, NKM-1,MEG-A2, NAGL-1, MTA, TMD5, KAI3, Kasumi-4, HL60(S), KHM-10B, K562/ADM,Kasumi-1, Kasumi-3, Kasumi-6, Ki-JK, CPT-K5, NOMO-1/ADM, NCO2, SKNO-1,KMS-11, KMM-1, KMS-21BM, KMS-26, KMS-27, KMS-28PE, KMS-28BM, KMS-34,KMS-20, TK, KCL-22, PL-21, MKPL-1, NALL-1, RC-K8, HD-70, DL-40,delta-47, PALL-2, FLAM-76, B104, KML-1, STR-428, KMS-33, KHM-2B,Kasumi-2, Kasumi-5, Kasumi-7, Kasumi-8, Kasumi-9, Kasumi-10, Minami-1,Minami-2, KasumiA-541, KasumiA-568, and KasumiA-554. The leukocytes orthe established cell line can be provided in a cryopreserved state.Examples of the cell culture medium include DMEM and RPMI 1640. The kitmay further comprise PHA that can be used as a positive control. The kitmay further comprise an agent for staining cells, preferably a reagentcapable of staining living cells (for example, POLARIC-500c6F), so thatthe cells can be easily observed under a microscope. In one embodiment,the present invention provides a device comprising a recorder forsequentially recording microscopic images of migration of cells and animage analyzer for analyzing the microscopic images to calculatemigration distance and migration velocity of the cells, for diagnosingan allergic disease in a subject; for determining a risk that a subjectdevelops an allergy; or for determining, detecting or quantifying anagent that is allergenic to a subject. The device may additionallycomprise the aforementioned kit. The kit and the device can be used inthe methods provided by the present invention.

The present invention further provides the following embodiments:

(a) A method for testing an allergy in an animal subject comprising,creating a test concentration gradient of body fluid collected from theanimal subject, and observing the cell kinetics of chemotactic cellsdepending on the presence or absence of a chemotactic factor containedin the body fluid or the concentration thereof in the test concentrationgradient.

(b) The method for testing an allergy in an animal subject according to(a), characterized in that a control concentration gradient is createdwith control body fluid collected from a control animal having noallergic symptom, the cell kinetics of the chemotactic cells dependingon the presence or absence of a chemotactic factor contained in thecontrol body fluid or the concentration thereof is observed in thecontrol concentration gradient, and the motility of the chemotacticcells in the test concentration gradient is compared with the motilityof the chemotactic cells in the control concentration gradient.

(c) A method for determining or testing an agent that is allergenic toan animal subject, which is for determining a causative agent for anallergy developed in an animal subject, in which a suspected agent thatis suspected to cause the allergy in the animal subject is mixed withbody fluid that contains at least lymphocytes and is collected from theanimal subject to give a test mixture, a test concentration gradient iscreated with the test mixture, and the cell kinetics of chemotacticcells depending on the presence or absence of a chemotactic factorcontained in the test mixture or the concentration thereof is observedin the test concentration gradient.

(d) The method for determining or testing an agent that is allergenic toan animal subject according to (c), in which body fluid that contains atleast lymphocytes and is collected from a control animal that has noallergy at least for the suspected agent is mixed with the suspectedagent to give a control mixture, a control concentration gradient iscreated with the control mixture, and the cell kinetics of chemotacticcells depending on the presence or absence of a chemotactic factorcontained in the control mixture or the concentration thereof isobserved in the control concentration gradient, and the motility of thechemotactic cells in the test concentration gradient is compared withthe motility of the chemotactic cells in the control concentrationgradient.

(e) A method for predicting an allergy in a subject, which is fordetermining or testing a causative agent for an allergy in an animalsubject before the animal subject develops an allergy, in which asuspected agent that is suspected to cause an allergy when used for ortaken by the animal subject is mixed with body fluid that contains atleast lymphocytes and is collected from the animal subject to give atest mixture, a test concentration gradient is created with the testmixture, and the cell kinetics of chemotactic cells depending on thepresence or absence of a chemotactic factor contained in the testmixture or the concentration thereof is observed in the testconcentration gradient.

(f) The method for predicting an allergy in a subject according to (e),in which body fluid that contains at least lymphocytes and is collectedfrom a control animal that has no allergy at least for the suspectedagent is mixed with the suspected agent to prepare a control mixture, acontrol concentration gradient is created with the control mixture, andthe cell kinetics of chemotactic cells depending on the presence orabsence of a chemotactic factor contained in the control mixture or theconcentration thereof is observed in the control concentration gradient,and the motility of the chemotactic cells in the test concentrationgradient is compared with the motility of the chemotactic cells in thecontrol concentration gradient.

In methods (a) to (f) described above, the animal subject and thecontrol animal may include both a human and a non-human animal. Theanimal subject and the control animal are preferably animals of the samespecies. Specifically, if the animal subject is a human, the controlanimal is preferably a human, and if the animal subject is a mouse, thecontrol animal is preferably a mouse.

In method (b) described above, the control animal having no allergicsymptom includes an animal having no allergic symptom and an animalhaving a slight allergy but having no subjective allergic symptom.

The present invention will be described in more detail with reference toexamples. The present invention is not limited to the following examplesat all. The examples are intended to illustrate the present inventionmore specifically. Modifications and changes that those skilled in theart can easily contemplate from the following examples shall be embracedwithin the scope of the present invention.

EXAMPLES Example 1: Detection of Drug Allergy Patient by UtilizingMigration of Leukocytes Derived from Healthy Human

(Patients to be Tested)

Blood withdrawal from patients having a drug allergy-like symptom andhealthy volunteers, and collection and use of leukocytes have beenapplied to and approved by the Institutional Review Board of FukuokaUniversity Hospital for a clinical study mainly using the LMT. Patientswho are candidates for the test were provided with explanation about theclinical study. Informed consent regarding the agreement and cooperationfor the clinical study was obtained in written form. Serum and reactionsolutions stimulated with antigens prepared for the LMT and remainedwere used, the LMT being for the patients who had developed a drugallergy-like symptom in the university hospital and needed determinationof the agent inducing the allergy-like symptom.

(Patient #1)

Hypersensitivity reaction: pancytopenia

Suspected drugs: Nafamostat mesilate (Naotamin), Nifedipine (Sepamit-R,Adalat-CR)

Clinical course: The pancytopenia was not improved after changing andwithdrawing the suspected drugs.

Accordingly, the symptom is unlikely to be caused by development of adrug allergy to the suspected drugs.

(Patient #2)

Hypersensitivity reaction: fever of unknown origin (39.0° C.)

Suspected drugs: Levofloxacin hydrate (Cravit), Doripenem hydrate(Finibax)

Clinical course: The patient had a fever of unknown origin before orafter administration of the suspected drugs. The fever naturally abated.

Accordingly, the symptom is very unlikely to be caused by development ofa drug allergy to the suspected drugs.

(Patient #3)

Hypersensitivity reaction: eosinophilia

Suspected drugs: Mosapride citrate hydrate (Gasmotin), Sulpride(Dogmatyl), Rabeprazole sodium (Pariet)

Clinical course: The eosinophils were reduced and the hypersensitivityreaction was improved after withdrawing the suspected drugs.

Accordingly, the symptom was strongly suspected to be caused by a drugallergy, and the causative agent is considered to be at least one of thesuspected drugs.

(Serum)

Blood samples from each subject (a healthy volunteer or a volunteer withmild cutaneous pruritus, and the patient suspected of having a drugallergy) were added with a serum separating agent and allowed to standat room temperature. Serum was separated by centrifugation at 2,000 rpmfor 30 minutes or by using Tube 21 (Registered Trademark) -S (SekisuiChemical Co., Ltd., Osaka).

(Chemotactic Cells)

Leukocytes including granulocytes such as neutrophils were separatedfrom heparinized blood of a healthy volunteer who had no allergicsymptom by the density gradient centrifugation (Lymphocyte (RegisteredTrademark) -poly: CEDARLANE Labs. Ltd., Ontario, Canada) in accordancewith a conventional method, and suspended in 5 ml of a culture medium(RPMI 1640: Sigma-Aldrich, MO, USA). After culturing the cells in a CO₂incubator at 37° C. for 1 hour the cells was adjusted to theconcentration of about 5×10⁵ cells/ml and used as chemotactic cells.

(Measurement of Cell Kinetics)

The chemotactic cells were injected into the lower well in the holder ofEZ-TAXIScan™ (GE Healthcare, Tokyo) at about 5 μl/well, and were drawnto the end of the observation terrace by suction. Subsequently, theserum was injected into the upper well at 1.5 μl/well with amicrosyringe to create a concentration gradient on the observationterrace, and images of the chemotactic cells on the observation terracewere sequentially recorded. The depth of the terrace was 4 μm.

(Analysis of Images)

The kinetics of the chemotactic cells migrating on the observationterrace was analyzed with software for processing images, ImageJ MD,USA). Various parameters concerned with chemotaxis or migration abilityof the cell toward the serum such as velocity, distance and direction ofthe migration were calculated. The statistical analysis was performedusing GraphPad Prism (Registered Trademark) (GraphPad Software, CA,USA).

The cell kinetics after the addition of 1.5 μl of the serum to the upperwell was analyzed using images recorded under microscope every twominutes for 20 minutes. Images obtained at the start and end of theobservation are shown in FIG. 1. The migration of the chemotactic cellson the observation terrace was successfully observed. FIG. 1 shows theimages obtained using the serum of the healthy volunteer (a), thepatients #1 to #3 (b to d), and the volunteer with cutaneous pruritus(e).

The polygonal lines on the observation terrace correspond to the tracksof the cells used in the analysis. In (a), (b), and (c), the migrationof the cells lacked direction and the migration distance was small. In(d) and (e), the movement of the cells was linear and the migrationdistance was longer than in (a), (b), and (c). Furthermore, in (d) and(e) the cells successively entered the observation terrace and manycells were on the observation terrace at the end of the analysis.

The cell migration of some selected cells was analyzed. FIG. 2 shows theresults. FIG. 2A shows the spatially coordinated migration of theindividual cells in reference to the original place where the cells wereat the time of starting the observation at the origin. FIGS. 2B and 2Cshow analytical results of the migration velocity and the migrationdistance, respectively. As expected, little change in both parameterswere seen for the healthy volunteer (a) and patients #1 and #2 (b and c)who are unlikely to have a drug allergy according to the clinicalcourses. On the other hand, for patient #3 (d) who is strongly suspectedto have a drug allergy and the volunteer (e) with mild cutaneouspruritus, the parameters were significantly different from those of allthe other groups.

(Criteria)

For setting criteria for the allergy test, percentages of the migrationvelocity and the migration distance relative to those of the controlgroup (the serum of the healthy volunteer: a) were calculated (FIGS. 3Aand 3B). When the criteria shown in Table 1, which classifies thedegrees of the allergy into four classes (strongly positive, ++:positive, +: suspected, +/−; and negative, −), was used, the results ofthe test were determined as shown in Table 2. Patient #3 was determinedas strongly positive (++) with either of the parameters, the migrationvelocity and the migration distance, the determination indicatingpatient #3 developed a strong allergy. Patient #1 and #2 were determinedas negative (−) in accordance with the clinical courses. The volunteerwith mild cutaneous pruritus was determined as positive or suspectedwith each parameter.

Compared to the actual symptom, the determination seems appropriate.Accordingly, setting and using such criteria is useful for an allergytest.

TABLE 1 Criterion (% of Control) <~120 120~150 150~200 >200 Judgment −+/− + ++

TABLE 2 Patient a b c d e Velocity − − − ++ +/− Distance − − − ++ +

Example 2: Detection of Drug Allergy Patient by

Utilizing Migration of Cells of Human T-Cell Cell Line

(Serum)

Serum was prepared in the substantially same manner as in Example 1.

(Chemotactic Cells)

In place of the leukocytes of the healthy volunteer, an establishedhuman T-cell cell line, Jurkat cell (American Type Culture Collection,VA, USA) was used as the chemotactic cells. The cells were suspended in5 ml of a culture medium (DMEM: Sigma-Aldrich), cultured in a CO₂incubator at 37° C. for 1 hour in the same manner as in Example 1,adjusted to the concentration of about 5×10⁵ cells/ml, and used as thechemotactic cells. The Jurkat cells were retained in a culture mediumfor subculture (DMEM containing 10% FBS (JRHBiosciences, KS, USA), 100U/ml penicillin and 100 μg/ml streptomycin (both Sigma-Aldrich)), withthe culture medium exchanged twice a week and, if necessary, theconcentration adjusted.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1 excepting that the depth of the terrace was set to 8 μm.

(Analysis of Images)

For determining the cell kinetics, the images were analyzed in thesubstantially same manner as in Example 1.

(Results) Compared to the cell kinetics of the human leukocytes used inExample 1, the cell kinetics of the Jurkat cells was very slow, themigration velocity was approximately ⅕. Accordingly, images recorded for50 minutes, rather than 20 minutes, were analyzed (FIGS. 4A and 4B).Similarly to Example 1, the migration velocity of measured for patient#3 (Example 1-d) was significantly larger than that that measured forthe healthy volunteer (Example 1-a) as shown in FIGS. 4C to 4E(p<0.001). Similar results were obtained for the migration distancethough the data is not shown. These results suggest that not onlyleukocytes containing granulocytes but also an appropriate establishedcell line may be used for the test, if it is suitable for the purpose ofthe test (the type of chemotactic factor that is necessary for the testor to be tested).

Example 3: Determination of Allergenic Agent by

The Method Provided by the Present Invention

(Serum)

Serum was prepared in the substantially same manner as in Example 1excepting that the subject was patient #3, who was found to clearly havea positive reaction in Example 1.

(Chemotactic Cells)

Chemotactic cells were prepared in the substantially same manner as inExample 1.

(Solution of Antigenic Drug)

A drug solution was prepared by dissolving a drug in a Hank's balancedsalt solution (HESS: Sigma-Aldrich) if the drug was a solid preparationsuch as a soluble tablet or a powder. If the drug was poorly soluble, itwas dissolved in Dimethyl-sulfoxide (DMSO: Sigma-Aldrich) and thesolution was diluted with HESS so that the final concentration of DMSOwas 1% or less. A solution containing the drug solution and the serum ofthe patient in a ratio of 1:1 was used as a solution of the antigenicdrug. The concentrations of the suspected drugs were set to ½ of theCmax in principle. The concentration of phytohemagglutinin (PHA: WakoPure Chemical Industries, Ltd., Osaka), a control antigen forstimulating or activating the lymphocytes, was set to 1 μg/ml.

(Mononuclear Cells)

Peripheral blood Mononuclear cells (PBMC) were separated fromheparinized blood of the patient by density gradient centrifugation(Lymphoprep™: Axis-Shield PoC, Oslo, Norway) in a conventional manner,washed with HBSS, and then adjusted to the concentration of about 5×10⁶cells/ml with a culture medium for reaction (RPMI 1640 containing 10%horse serum (Invitrogen, CA, USA)).

(Reactive Solution Stimulated with Antigen)

A mixture of the suspension of the mononuclear cells and the solution ofthe antigenic drug in a ratio of 4:1 was reacted in a CO₂ incubator at37° C. for 72 hours, and the supernatant was used as a reactive solutionstimulated with the antigen. PHA (a), gasmotin (b), dogmatyl (c), pariet(d) and DMSO (e) were used as the antigen.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1 excepting that the reactive solution stimulated with theantigen was injected into the upper well in place of the serum.

(Analysis of Images)

The images were analyzed in the substantially same manner as in Example1.

(Results)

Since human leukocytes were used, images recorded for 20 minutes wereused for the analysis as in Example 1. As shown in FIG. 5A, whengasmotin (b) was used as the antigen, rapid migration of the chemotacticcells in the positive direction of the density gradient of the reactivesolution stimulated with the antigen, which is similar to that observedwith the serum, was observed on the observation terrace. PHA (a) as thecontrol induced the chemotaxis or migration ability weaker than thatinduced with (b) but stronger than those induced with the othersuspected drugs (c and d) or the solvent (DMSO, e). The cell migrationof some selected cells was analyzed in detail. The migration velocity(FIG. 5B) and migration distance (FIG. 5C) induced with the suspecteddrug b was significantly larger than that induced with the othersuspected drugs and the solvent (c, d and e). The results stronglysuggest that drug b (gasmotin) induced the drug allergy in the patient#3. The symbol “*” in FIGS. 5B and 5C indicates the significantdifference from the group of the suspected drug b (p<0.05).

(Criteria)

For setting criteria for determining the causative agent that inducesthe drug allergy or for predicting the allergy in a patient, percentagesof the migration velocity and the migration distance relative to that ofthe control group (the reactive solution stimulated with PHA: a) werecalculated (FIGS. 6A and 6B). When the criteria shown in Table 3, whichclassifies the degrees of the allergy into four classes (stronglypositive, ++: positive, +: suspected, +/−; and negative, −), is used,the results of the test are determined as shown in Table 4. Among thesuspected drugs for Patient #3, gasmotin (b) was determined as stronglypositive (++) with either of the parameters, the migration velocity andthe migration distance, the determination indicating that gasmotin (b)was the causative agent inducing the drug allergy. The other suspecteddrugs and the solvent (c, d and e) were determined as suspected (+/−) ornegative (−) particularly on the basis of the migration distance. Sincethe large change in the chemotaxis of leukocytes was observed in thisexample by posteriorly stimulating the lymphocytes of the patient havingthe drug allergy with the drug and the serum, it will be generallypossible to determine development of a drug allergy by posteriorly orpreviously stimulating lymphocytes with the drug. Accordingly, settingand using such criteria is useful for determining a causative agent ofan allergy or predicting a drug allergy in a patient.

TABLE 3 Criterion (% of Control) <~80 80~90 90~120 >120 Judgment − +/− +++

TABLE 4 Patient a b c d e Velocity + ++ +/− + − Distance + ++ +/− +/− −

Example 4: Influence of Antigen Concentration on Cell Kinetics ofMigrating Cells

(Serum)

Serum was prepared in the substantially same manner as in Example 1excepting that the subject was a volunteer who had a medical history ofa cedar pollen allergy but did not develop an allergic symptom at thetime of the blood withdrawal (September).

(Chemotactic Cells)

Chemotactic cells were prepared in the substantially same manner as inExample 1.

(Solution of Antigenic Drug)

A cedar pollen antigen SBP (Hayashibara Biochemical Laboratories, Inc.,Okayama) was used. The serum was prepared so that the finalconcentration is 10%.

(Peripheral Blood Mononuclear Cells)

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized blood of the volunteer having the medical history of thecedar pollen allergy but not developing the symptom at the time of bloodwithdrawal by density gradient centrifugation in a conventional manner,washed with PBS, and then adjusted to the concentration of about 5×10⁶cells/ml with a culture medium for reaction (RPMI 1640 containing 10%FES).

(Reactive Solution Stimulated with Antigen)

The solutions of the antigenic drug (the cedar pollen antigen SBP: 0,0.5, 5, or 50 μg/ml) were added to the mononuclear cells, the mixtureswere reacted in a CO₂ incubator at 37° C. for 72 hours, and thesupernatants were used as the reactive solution stimulated with theantigen.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1 excepting that the reactive solutions stimulated with theantigen was injected into the upper well in place of the serum.

(Analysis of Images)

The images were analyzed in the substantially same manner as in Example1.

(Results)

In the same manner as in Example 3, the activation and migration of thechemotactic cells in the positive direction of the density gradient ofthe reactive solution stimulated with the antigen, the cedar pollenantigen SBP, was observed on the observation terrace (FIG. 7A). Thecells were activated depending on the concentration of the antigen. Thecell migration of some selected cells was analyzed in detail. Asexpected, the migration velocity (FIG. 7B) and the migration distance(FIG. 7C) significantly increased depending on the concentration of theantigen (*: p<0.01, †: p<0.001).

(Criteria)

For setting criteria for predicting development of the allergy,percentages of the migration velocity and the migration distancerelative to that of the control group (the cedar pollen antigen SEP=0μg/ml) were calculated (FIGS. 8A and 8E). When the criteria shown inTable 5, which classifies the degrees of the allergy into four classes(strongly positive, ++: positive, +: suspected, +/−; and negative, −),is used, the results of the test are determined as shown in Table 6. Thesubject was determined as strongly positive (++) or suspected (+/−) witheither of the parameters, the determination indicating that the pollenallergy was induced. The determination can predict that the subject isvery likely to develop a serious pollen allergy when the subject isexposed to cedar pollen in future. Accordingly, it is suggested that anovel method for predicting development of an allergy in the absence ofan allergic symptom can be provided, the method using a reactivesolution stimulated with an antigen which is prepared by stimulatinglymphocytes and serum of a subject with an antigen, determining andanalyzing chemotaxis or migration ability of chemotactic cells, andapplying an appropriate criteria to the result.

TABLE 5 Criterion (% of Control) <~120 120~140 140~160 >160 Judgment −+/− + ++

TABLE 6 Conc. (μg/ml) 0 0.5 5.0 50 Velocity − +/− +/− ++ Distance − ++++ ++

Example 5: Influence of Stimulation with Pollen Extract to Cell Kineticsin the Presence or Absence of Pollen Allergy in Subject

(Chemotactic Cells)

Chemotactic cells were prepared in the substantially same manner as inExample 1.

(Solution of Antigenic Drug)

A cedar pollen antigen SBP (Hayashibara Biochemical Laboratories, Inc.,Okayama) was used. The serum was prepared so that the finalconcentration is 10%.

(Peripheral Blood Mononuclear Cells)

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized blood of a patient having a cedar pollen allergy and ahealthy volunteer having no pollen allergy by density gradientcentrifugation in a conventional manner, washed with PBS, and thenadjusted to the concentration of about 5×10⁶ cells/ml with a culturemedium for reaction (RPMI 1640 containing 10% FES).

(Reactive Solution Stimulated with Antigen)

The solutions of the antigenic drug (the cedar pollen antigen SBP: 0,0.5, 5, or 50 μg/ml) were added to the mononuclear cells, the mixtureswere reacted in a CO₂ incubator at 37° C. for 72 hours, and thesupernatants were used as the reactive solution stimulated with theantigen.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1 excepting that the reactive solutions stimulated with theantigen were injected into the upper well in place of the serum.

(Image Analysis)

The images were analyzed in the substantially same manner as in Example1.

In the test using the mononuclear cells exposed to the pollen extract,cell kinetics of the leukocytes of a healthy volunteer was clearlydifferent between the subject having the pollen allergy and the subjecthaving no pollen allergy. When the subject had no pollen allergy, thecell kinetics of the leukocytes of the healthy volunteer was notaffected at all by the supernatant of the mononuclear cells stimulatedwith the pollen extract. On the other hand, when the subject had apollen allergy, the motility of the leukocytes of the healthy volunteercaused by the supernatant of the stimulated mononuclear cells increaseddepending on the concentration of the pollen extract (FIG. 9) (*:p<0.01). This result indicates that mononuclear cells of a patient of apollen allergy secretes upon stimulation with pollen a chemotacticfactor which is different from those of a healthy human, and that thechemotactic factor causes migration of leukocytes of a healthy human.

Example 6: Diagnosis of Subject Having Allergy to Kampo Product andDetermination of Allergenic Agent by Method Provided by the PresentInvention

(Chemotactic Cells)

Chemotactic cells were prepared in the substantially same manner as inExample 1.

(Solution of Antigenic Drug)

To 550 ml of purified water, 18 g of a Kampo product (Nagakura Hinyosen)was added, and the mixture was heated and concentrated to the volume of330 ml, filtered through a paper filter to give a decoction of the Kampoproduct. Decoctions of four components of the Kampo product, Senkotsu(Nuphar japonicum), Byakujutsu (Atractylodes japonica), Bukuryo(Wolfiporia extensa) and Mokutsu (Akebia quinata), were also prepared.

(Peripheral Blood Mononuclear Cells)

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized blood of a patient having a cedar pollen allergy and ahealthy volunteer having no pollen allergy by density gradientcentrifugation in a conventional manner, washed with PBS, and thenadjusted to the concentration of about 5×10⁶ cells/ml with a culturemedium for reaction (RPMI 1640 containing 10% FBS).

(Reactive Solution Stimulated with Antigen)

A mixture of the mononuclear cells and the solution of the antigenicdrug was reacted in a CO₂ incubator at 37° C. for 72 hours, and thesupernatant was used as a reactive solution stimulated with the antigen.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1 excepting that the reactive solution stimulated with theantigen was injected into the upper well in place of the serum.

(Analysis of Images)

The images were analyzed in the substantially same manner as in Example1.

The mononuclear cells of a patient, who had an allergy-like symptomafter taking the Kampo product (Nagakura Hinyosen), were exposed to thesuspected drug, the decoction of the Kampo product (1:10,000), and theinfluence of the culture supernatant on the cell kinetics of theleukocytes of a healthy volunteer was examined. The exposure of themononuclear cells of the patient to the Kampo product induced productionand release of a chemotactic factor from the cells, and the chemotacticfactor affected the cell kinetics of the leukocytes of the healthyvolunteer. The result suggests that the subject has developed an allergy(FIG. 10A) (*: p<0.01).

Decoctions of each component of the Kampo product (senkotsu, byakujutsu,bukuryo and mokutsu) were prepared and tested. Senkotsu and byakujutsuinduced very strong reactions similar to that of the Hinyosen. Thesubject is thought to have a strong allergic reaction to these twocomponents of the Kampo product. The result suggests that the subjectshould avoid taking a product such as a medicine containing thesecomponents (FIG. 10B) (*: p<0.01).

Example 7: Comparison Between Diagnosis by Method Provided by thePresent Invention and Diagnosis by DLST or LMT

(Serum)

Blood samples from a patient who developed a typical allergy-likesymptom and a patient with no such symptom were added with a serumseparating agent and allowed to stand at room temperature. Serum wasseparated by centrifugation at 2,000 rpm for 30 minutes or by using Tube21 (Registered Trademark) -S (Sekisui Chemical Co., Ltd., Osaka).

(Chemotactic Cells)

Chemotactic cells were prepared in the substantially same manner as inExample 1.

(Solution of Antigenic Drug)

A drug solution was prepared by dissolving a drug in a Hank's balancedsalt solution (HESS: Sigma-Aldrich) if the drug was a solid preparationsuch as a soluble tablet or a powder. If the drug was poorly soluble, itwas dissolved in Dimethyl-sulfoxide (DMSO: Sigma-Aldrich) and thesolution was diluted with HESS so that the final concentration of DMSOwas 1% or less. A solution containing the drug solution and the serum ofthe patient in a ratio of 1:1 was used as a solution of the antigenicdrug. The concentrations of the suspected drugs were set to ½ of theCmax in principle. The concentration of phytohemagglutinin (PHA: WakoPure Chemical Industries, Ltd., Osaka), a control antigen forstimulating or activating lymphocytes, was set to 1 μg/ml.

(Antigen Stimulation Reaction Solution)

A mixture of the mononuclear cells and the solution of the antigenicdrug was reacted in a CO₂ incubator at 37° C. for 72 hours, and thesupernatant was used as a reactive solution stimulated with an antigen.

(Peripheral Blood Mononuclear Cells)

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized blood of the patient who developed the typical allergy-likesymptom and the patient with no such symptom by density gradientcentrifugation in a conventional manner, washed with PBS, and thenadjusted to the concentration of about 5×10⁶ cells/ml with a culturemedium for reaction (RPMI 1640 containing 10% FBS).

(Measurement of Cell Kinetics)

The cell kinetics was measured in the substantially same manner as inExample 1.

(Analysis of Images)

The images were analyzed in the substantially same manner as in Example1.

The patient having the typical allergy-like symptom (skin lesion) andthe atypical patient were tested by the three different methods, namelythe DLST, which is widely used for clinical tests, the LMT, which issometimes reported more accurate than the DLST, and the present method,and then the results were compared. As shown in Table 7, the atypicalcase (patient A) and the typical case (patient B) were determined asnegative by the DLST and the LMT, which are currently employed as theprimary testing methods. The typical case was determined as negativethough the patient clearly developed the skin symptom of the toxicepidermal necrolysis (TEN) according to the clinical presentation andthe clinical course.

On the other hand, the method provided by the present inventiondetermined that the cell kinetics of the atypical case was negative forall the suspected drugs excluding PHA, the positive control, providingthe same result as those of the DLST and the LMT (FIG. 11) (*: p<0.01).These results suggest that the symptom of the atypical patient wascaused by a cause other than the allergy.

The method provided by the present invention, unlike the DLST and theLMT, determined that the two suspected drugs as well as the oralpreparations containing the drugs were positive in the typical case(FIG. 12) (*: p<0.01), Accordingly, the method provided by the presentinvention revealed that the suspected drug (the medical ingredient)caused the development of the TEN.

TABLE 7 Patient Patient A Patient B Suspected drug Itrizole ItraconazolePHA Maxipime Zosyn PHA test 1 test 2 test 1 test 2 DLST (SI value)* +112 124 + 100 100 151 90 LMT (MI value)# 154.87 88.21 98.42 283.9 ND100.7 ND 135.5 Positive with *SI value > 180 (DLST), #MI value > 150 andp < 0.05 (LMT); ND, not determined.

Example 8: Detection of Allergenic Agent Using Human Promyelocytic CellLine: HL-60 Cells

(Serum)

Serum of a healthy human was prepared in the same manner as in Example1.

(Chemotactic Cells)

A human promyleocytic cell line: HL-60 cells were used as chemotacticcells. The cells were suspended in 5 ml of a culture medium (RPMI 1640),cultured in a CO₂ incubator at 37° C. for 1 hour, adjusted to theconcentration of about 5×10⁵ cells/ml, and used as the chemotacticcells. The HL-60 cells were retained in a culture medium for subculture(DMEM containing 10% FBS (JRHBiosciences, KS, USA), 100 U/ml penicillinand 100 μg/ml streptomycin (bath Sigma-Aldrich)), with the culturemedium exchanged and, if necessary, the concentration adjusted twice aweek.

(Solution of Antigenic Drug)

A cedar pollen antigen SEP (Hayashibara Biochemical Laboratories, Inc.,Okayama) was used. The concentration was adjusted to be at 50 μg/mlafter the addition of the antigen to the mononuclear cells.

(Peripheral Blood Mononuclear Cell)

Peripheral blood mononuclear cells (PBMC) were separated fromheparinized blood of a patient having a cedar pollen allergy by densitygradient centrifugation in a conventional manner, washed with PBS, andthen adjusted to the concentration of about 5×10⁶ cells/ml with aculture medium for reaction (RPMI 1640 containing 10% FBS).

(Reactive Solution Stimulated with Antigen)

Serum of a healthy human or a pollen extract diluted with serum of thehealthy human was added to the mononuclear cells, the mixtures werereacted in a CO₂ incubator at 37° C. for 72 hours, and the supernatantswere used as the reactive solution stimulated with the antigen.

(Measurement of Cell Kinetics)

The cell kinetics was measured in the same manner as in Example 4.

(Analysis of Images)

The images were analyzed in the same manner as in Example 4.

The culture supernatant of the mononuclear cells of the patient having acedar pollen allergy that was exposed to the pollen extract increasedthe migration distance of the HL-60 cells (FIG. 13). The resultindicates that the mononuclear cells of the patient having a cedarpollen allergy secreted a chemotactic factor which is not present in theserum of the healthy human after the exposure to the pollen extract, andthat the chemotactic factor affects the cell kinetics of the HL-60cells.

1-14. (canceled)
 15. A method for determining whether a sample contains an agent that is allergenic to a subject who developed an allergy, comprising the steps of: (1) obtaining leukocytes from the subject; (2) culturing the leukocytes in a medium containing the sample; (3) collecting the culture supernatant; (4) creating a concentration gradient of the culture supernatant; (5) placing chemotactic cells in the concentration gradient of the culture supernatant; and (6) measuring the migration distance and/or migration velocity of the chemotactic cells, wherein the chemotactic cells are leukocytes derived from a healthy animal or cells of leukocytic cell line, and wherein (a) steps (2)-(6) are further carried out by using leukocytes derived from a healthy animal in place of the leukocytes from the subject and phytohemagglutinin in place of the sample, (i) the sample is determined to contain an agent that is allergenic to the subject when the migration distance of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the subject and the sample is equivalent to or longer than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the healthy animal and phytohemagglutinin; and/or (ii) the sample is determined to contain an agent that is allergenic to the subject when the migration velocity of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the subject and the sample is equivalent to or higher than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the healthy animal and phytohemagglutinin, or (b) steps (2) to (6) are also carried out by using phytohemagglutinin as a positive control in place of the sample, and (i) the sample is determined to contain an agent that is allergenic to the subject when the migration distance of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the sample is equivalent to or longer than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the phytohemagglutinin; and/or (ii) the sample is determined to contain an agent that is allergenic to the subject when the migration velocity of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the sample is equivalent to or higher than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the phytohemagglutinin.
 16. The method according to claim 15, wherein (a) steps (2)-(6) are further carried out by using leukocytes derived from a healthy animal in place of the leukocytes from the subject and phytohemagglutinin in place of the sample, (i) the sample is determined to contain an agent that is allergenic to the subject when the migration distance of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the subject and the sample is equivalent to or longer than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the healthy animal and phytohemagglutinin; and/or (ii) the sample is determined to contain an agent that is allergenic to the subject when the migration velocity of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the subject and the sample is equivalent to or higher than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the leukocytes from the healthy animal and phytohemagglutinin.
 17. The method according to claim 15, wherein (b) steps (2) to (6) are also carried out by using phytohemagglutinin as a positive control in place of the sample, and (i) the sample is determined to contain an agent that is allergenic to the subject when the migration distance of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the sample is equivalent to or longer than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the phytohemagglutinin; and/or (ii) the sample is determined to contain an agent that is allergenic to the subject when the migration velocity of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the sample is equivalent to or higher than that of the chemotactic cells placed in the concentration gradient of the culture supernatant prepared with the phytohemagglutinin.
 18. The method according to claim 15, wherein the allergy is a pollen allergy, a chemical allergy, a drug allergy or a food allergy.
 19. The method according to claim 15, wherein the sample comprises pollen, a chemical, a drug or a food.
 20. The method according to claim 15, wherein the chemotactic cells are cells of a T-cell cell line or a promyelocytic cell line.
 21. The method according to claim 15, wherein the chemotactic cells are Jurkat cells or HL-60 cells.
 22. The method according to claim 15, wherein the migration of the chemotactic cells is sequentially recorded over time, and the migration distance or the migration velocity of the cells is determined on the basis of the record over time.
 23. The method according to claim 15, wherein the subject is a mammal.
 24. The method according to claim 15, wherein the subject is a human.
 25. The method according to claim 23, wherein the chemotactic cells are leukocytes derived from a healthy mammal.
 26. The method according to claim 24, wherein the chemotactic cells are leukocytes derived from a healthy human.
 27. The method according to claim 23, wherein the chemotactic cells are cells of leukocytic cell line.
 28. The method according to claim 24, wherein the chemotactic cells are cells of leukocytic cell line. 